The overall goal of this project is to determine the mechanism(s) leading to excess cholesterol accumulation in macrophages treated with apolipoprotein E4 (ApoE4)-enriched lipoproteins, and to elucidate the role of unfolded protein response (UPR) in this process. There are three major human alleles of apolipoprotein 5 (Apo5), i.e., 52, 53, and 54. The 54 product, ApoE4, is associated with increased risk of atherosclerotic cardiovascular disease compared to ApoE3, the so-called normal isoform. Apo54 is prevalent in African-Americans, for whom cardiovascular disease is disproportionally high compared to other ethnic groups. Previous work in Dr. ZhongMao Guo"s laboratory demonstrated that increased eukaryotic initiation factor 21 (eIF-21) phosphorylation, a signaling of UPR, is a mechanism underlying cholesterol accumulation and foam cell formation in macrophages treated with lipoproteins derived from ApoB48/48/ApoE-/- (E>/B48) mice. Recently, we found that E>/B48 lipoproteins enriched with human ApoE4 (E4/B48) induce greater cholesterol accumulation and enhanced eIF-21 phosphorylation in macrophages than an equal concentration of E-/B48 lipoproteins enriched with the same amount of human ApoE3 (E3/B48). In addition, E4/B48 lipoproteins are less able than E3/B48 lipoproteins to induce the expression of the cholesterol efflux transporter, ATP-binding cassette transporter A1 (ABCA1), in macrophages. Deficiency of this transporter is associated with widespread foam cell formation and severe atherosclerosis. This project will test the hypothesis that activation of UPR pathways is a mechanism behind the reduction in transporter expression mediating cholesterol efflux, leading to accumulation of excess cholesterol in macrophages treated with E4/B48 lipoproteins. Two specific aims will be carried out to test this hypothesis. Specific Aim 1 will study the effect of E4/B48-lipoproteins on macrophage expression of transporters mediating cholesterol efflux, cholesterol efflux itself, and foam cell formation. This aim will be accomplished by measuring the expression levels of cholesterol efflux transporters (ABCA1, ABCG1, and scavenger receptor-B1 [SR-B1]), evaluating the degree of cholesterol efflux, and determining the extent of cellular cholesterol accumulation after macrophages are treated with E4/B48 and E3/B48 lipoproteins. Specific Aim 2 will determine the role that UPR signaling pathways play in macrophage expression of transporters mediating cholesterol efflux, cholesterol efflux itself, and foam cell formation. There are three UPR pathways. This project will focus on the eIF21-associated pathway because E4/B48 lipoproteins have been shown to activate it. If our hypothesis is correct, changes in expression of cholesterol efflux transporters, and cholesterol efflux and accumulation induced by E4/B48 lipoproteins will be attenuated when this pathway is inhibited. PUBLIC HEALTH RELEVANCE: There are three major human alleles of apolipoprotein 5 (Apo5), i.e., 52, 53, and 54, and the 54 isoform is associated with increased risk of atherosclerotic cardiovascular disease compared to the normal 53. African-Americans have higher 54 allelic frequency and are more prone to cardiovascular diseases related to atherosclerosis compared to other ethnic groups. By elucidating the role that ApoE4-containing lipoproteins play in the formation of foam cells, an early event in the development of atherosclerosis, we will provide strategies for treatment or prevention of this disease.